Plasmodesmata dynamics in bryophyte model organisms: secondary formation and developmental modifications of structure and function.

MAIN CONCLUSION: Developing bryophytes differentially modify their plasmodesmata structure and function. Secondary plasmodesmata formation via twinning appears to be an ancestral trait. Plasmodesmata networks in hornwort sporophyte meristems resemble those of angiosperms. All land-plant taxa use plasmodesmata (PD) cell connections for symplasmic communication. In angiosperm development, PD networks undergo an extensive remodeling by structural and functional PD modifications, and by postcytokinetic formation of additional secondary PD (secPD). Since comparable information on PD dynamics is scarce for the embryophyte sister groups, we investigated maturating tissues of Anthoceros agrestis (hornwort), Physcomitrium patens (moss), and Marchantia polymorpha (liverwort). As in angiosperms, quantitative electron microscopy revealed secPD formation via twinning in gametophytes of all model bryophytes, which gives rise to laterally adjacent PD pairs or to complex branched PD. This finding suggests that PD twinning is an ancient evolutionary mechanism to adjust PD numbers during wall expansion. Moreover, all bryophyte gametophytes modify their existing PD via taxon-specific strategies resembling those of angiosperms. Development of type II-like PD morphotypes with enlarged diameters or formation of pit pairs might be required to maintain PD transport rates during wall thickening. Similar to angiosperm leaves, fluorescence redistribution after photobleaching revealed a considerable reduction of the PD permeability in maturating P. patens phyllids. In contrast to previous reports on monoplex meristems of bryophyte gametophytes with single initials, we observed targeted secPD formation in the multi-initial basal meristems of A. agrestis sporophytes. Their PD networks share typical features of multi-initial angiosperm meristems, which may hint at a putative homologous origin. We also discuss that monoplex and multi-initial meristems may require distinct types of PD networks, with or without secPD formation, to control maintenance of initial identity and positional signaling.

Multicellularity and the Need for Communication-A Systematic Overview on (Algal) Plasmodesmata and Other Types of Symplasmic Cell Connections.

In the evolution of eukaryotes, the transition from unicellular to simple multicellular organisms has happened multiple times. For the development of complex multicellularity, characterized by sophisticated body plans and division of labor between specialized cells, symplasmic intercellular communication is supposed to be indispensable. We review the diversity of symplasmic connectivity among the eukaryotes and distinguish between distinct types of non-plasmodesmatal connections, plasmodesmata-like structures, and 'canonical' plasmodesmata on the basis of developmental, structural, and functional criteria. Focusing on the occurrence of plasmodesmata (-like) structures in extant taxa of fungi, brown algae (Phaeophyceae), green algae (Chlorophyta), and streptophyte algae, we present a detailed critical update on the available literature which is adapted to the present classification of these taxa and may serve as a tool for future work. From the data, we conclude that, actually, development of complex multicellularity correlates with symplasmic connectivity in many algal taxa, but there might be alternative routes. Furthermore, we deduce a four-step process towards the evolution of canonical plasmodesmata and demonstrate similarity of plasmodesmata in streptophyte algae and land plants with respect to the occurrence of an ER component. Finally, we discuss the urgent need for functional investigations and molecular work on cell connections in algal organisms.

Pea Aphid (Acyrthosiphon pisum) Host Races Reduce Heat-Induced Forisome Dispersion in Vicia faba and Trifolium pratense.

Although phloem-feeding insects such as aphids can cause significant damage to plants, relatively little is known about early plant defenses against these insects. As a first line of defense, legumes can stop the phloem mass flow through a conformational change in phloem proteins known as forisomes in response to Ca2+ influx. However, specialized phloem-feeding insects might be able to suppress the conformational change of forisomes and thereby prevent sieve element occlusion. To investigate this possibility, we triggered forisome dispersion through application of a local heat stimulus to the leaf tips of pea (Pisum sativum), clover (Trifolium pratense) and broad bean (Vicia faba) plants infested with different pea aphid (Acyrthosiphon pisum) host races and monitored forisome responses. Pea aphids were able to suppress forisome dispersion, but this depended on the infesting aphid host race, the plant species, and the age of the plant. Differences in the ability of aphids to suppress forisome dispersion may be explained by differences in the composition and quantity of the aphid saliva injected into the plant. Various mechanisms of how pea aphids might suppress forisome dispersion are discussed.

Extracellular vesicles isolated from dsRNA-sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum.

Numerous reports have shown that incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto their leaves successfully protects them against invading pathogens exploiting the mechanism of RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown. It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their pathogens. Recently, we found that EVs isolated from host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS) plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from dsRNA-sprayed barley (Hordeum vulgare) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum. Encouraged by our previous finding that dropping barley-derived EVs on F. graminearum cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated F. graminearum with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of F. graminearum macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect F. graminearum growth and showed no gene silencing activity on F. graminearum CYP51 genes. Based on our findings, we concluded that either the amount of SIGS-derived siRNA was insufficient to induce target gene silencing in F. graminearum, indicating that the role of EVs in SIGS is minor, or that F. graminearum uptake of plant EVs from liquid cultures was inefficient or impossible.

Developmental Plasticity of the Amphibious Liverwort Riccia fluitans.

The colonization of land by ancestors of embryophyte plants was one of the most significant evolutionary events in the history of life on earth. The lack of a buffering aquatic environment necessitated adaptations for coping with novel abiotic challenges, particularly high light intensities and desiccation as well as the formation of novel anchoring structures. Bryophytes mark the transition from freshwater to terrestrial habitats and form adaptive features such as rhizoids for soil contact and water uptake, devices for gas exchange along with protective and repellent surface layers. The amphibious liverwort Riccia fluitans can grow as a land form (LF) or water form (WF) and was employed to analyze these critical traits in two different habitats. A combination of light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies was conducted to characterize and compare WF and LF morphologies. A complete phenotypic adaptation of a WF plant to a terrestrial habitat is accomplished within 15 days after the transition. Stable transgenic R. fluitans lines expressing GFP-TUBULIN and mCherry proteins were generated to study cell division and differentiation processes and revealed a higher cell division activity in enlarged meristematic regions at LF apical notches. Morphological studies demonstrated that the R. fluitans WF initiates air pore formation. However, these pores are arrested at an early four cell stage and do not develop further into open pores that could mediate gas exchange. Similarly, also arrested rhizoid initial cells are formed in the WF, which exhibit a distinctive morphology compared to other ventral epidermal cells. Furthermore, we detected that the LF thallus has a reduced surface permeability compared to the WF, likely mediated by formation of thicker LF cell walls and a distinct cuticle compared to the WF. Our R. fluitans developmental plasticity studies can serve as a basis to further investigate in a single genotype the molecular mechanisms of adaptations essential for plants during the conquest of land.

Species-Specific and Distance-Dependent Dispersive Behaviour of Forisomes in Different Legume Species.

Forisomes are giant fusiform protein complexes composed of sieve element occlusion (SEO) protein monomers, exclusively found in sieve elements (SEs) of legumes. Forisomes block the phloem mass flow by a Ca2+-induced conformational change (swelling and rounding). We studied the forisome reactivity in four different legume species-Medicago sativa, Pisum sativum, Trifolium pratense and Vicia faba. Depending on the species, we found direct relationships between SE diameter, forisome surface area and distance from the leaf tip, all indicative of a developmentally tuned regulation of SE diameter and forisome size. Heat-induced forisome dispersion occurred later with increasing distance from the stimulus site. T. pratense and V. faba dispersion occurred faster for forisomes with a smaller surface area. Near the stimulus site, electro potential waves (EPWs)-overlapping action (APs), and variation potentials (VPs)-were linked with high full-dispersion rates of forisomes. Distance-associated reduction of forisome reactivity was assigned to the disintegration of EPWs into APs, VPs and system potentials (SPs). Overall, APs and SPs alone were unable to induce forisome dispersion and only VPs above a critical threshold were capable of inducing forisome reactions.

Comparison of intracellular location and stimulus reaction times of forisomes in sieve tubes of four legume species.

Forisomes in legumes are responsible for fast sieve-element occlusion in response to injury to the vascular system. This prevents uncontrolled leakage of phloem sap and protects against invasion of pathogens. Here we compared forisomes of four different legumes (Pisum sativum, Vicia faba, Trifolium pratense and Medicago sativa) by their location (basal, central, apical) in the sieve element and reactivity to a distant heat stimulus. In each species, the majority of forisomes was located basally. Yet, we found differences in intracellular location: forisomes are distributed more evenly in the sieve elements of Pisum. After burning, basally located forisomes of the four species reacted with dispersion, followed by a spontaneous recondensation with similar reaction times. The results suggest universal forisome behaviour in fabacean species.